Recombinant Vibrio vulnificus Holo-[acyl-carrier-protein] synthase (acpS)

What is the role of Holo-[acyl-carrier-protein] synthase (acpS) in Vibrio vulnificus metabolism?

Holo-[acyl-carrier-protein] synthase (acpS) plays a critical role in Vibrio vulnificus metabolism by catalyzing the conversion of inactive apo-ACP to active holo-ACP through the transfer of a 4'-phosphopantetheine moiety from coenzyme A to a conserved serine residue on the acyl carrier protein. This post-translational modification is essential for fatty acid biosynthesis, as the activated holo-ACP serves as the primary carrier of acyl intermediates during fatty acid chain elongation . In V. vulnificus, this process is particularly important for membrane phospholipid biosynthesis, which contributes to the organism's cellular integrity and, indirectly, to its virulence potential. The acpS enzyme represents a critical junction point in metabolic regulation, connecting coenzyme A metabolism with fatty acid and phospholipid biosynthetic pathways that support the growth and pathogenicity of this opportunistic pathogen.

How does V. vulnificus acpS differ structurally from other bacterial acpS enzymes?

V. vulnificus acpS shares the core catalytic domain structure with other bacterial acpS enzymes but exhibits species-specific sequence variations that may affect substrate specificity and regulatory interactions. While the active site architecture maintains the conserved residues necessary for CoA binding and phosphopantetheinyl transfer activity, V. vulnificus acpS contains unique surface residues that potentially interact with regulatory proteins specific to Vibrio species . These structural differences may reflect adaptations to the brackish water environment where V. vulnificus naturally resides and could influence how the enzyme functions under varying salt concentrations and temperatures. Notably, comparative structural analyses suggest that V. vulnificus acpS may contain distinctive binding interfaces for interaction with the FadR regulatory protein, which has been shown to regulate fatty acid metabolism genes in Vibrio species . These structural adaptations likely contribute to the specialized regulatory networks that control membrane composition in this pathogen.

What experimental evidence connects acpS function to V. vulnificus virulence?

While direct experimental evidence linking acpS function to V. vulnificus virulence is limited in the current literature, several indirect connections can be established. Research has demonstrated that V. vulnificus virulence strongly correlates with capsular polysaccharide (CPS) expression , and proper membrane composition is essential for CPS attachment and presentation. Since acpS is crucial for fatty acid biosynthesis and, consequently, membrane phospholipid composition, its activity likely influences CPS expression and arrangement. Studies have shown that encapsulated V. vulnificus with opaque colony morphologies exhibit enhanced virulence compared to translucent variants with reduced CPS . The spacing and organization of outer membrane vesicles (OMVs), which are affected by membrane composition, also differ between virulent encapsulated strains and less virulent unencapsulated variants . Because acpS function is central to maintaining proper membrane structure, it likely plays an indirect but significant role in these virulence-associated phenotypes. Future research specifically targeting acpS activity through conditional mutants could more directly establish its contribution to virulence mechanisms.

What are the optimal conditions for expressing recombinant V. vulnificus acpS in E. coli expression systems?

The optimal conditions for expressing recombinant V. vulnificus acpS in E. coli systems require careful consideration of several parameters. For maximal protein yield and activity, expression should be conducted in BL21(DE3) or Rosetta(DE3) strains harboring the pET expression system with the acpS gene optimized for E. coli codon usage. Induction with 0.5 mM IPTG at mid-log phase (OD600 = 0.6-0.8) and subsequent growth at 25°C for 6-8 hours typically yields better results than standard 37°C expression, as lower temperatures reduce inclusion body formation . The addition of 2% glucose to the culture medium helps suppress basal expression before induction. For buffer optimization, the enzyme shows highest stability in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10% glycerol, and 1 mM DTT. Media supplementation with 1 mM MgCl2 has been observed to enhance the catalytic activity of the purified enzyme. When scaling up production, maintaining dissolved oxygen levels above 30% saturation throughout the fermentation process is critical for achieving consistent enzyme activity across batches.

What purification strategy yields the highest specific activity for recombinant V. vulnificus acpS?

A multi-step purification strategy is required to obtain recombinant V. vulnificus acpS with high specific activity. The optimal protocol begins with cell lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 1 mM PMSF using sonication (10 cycles of 30 seconds on/off at 40% amplitude). After centrifugation at 15,000 × g for 30 minutes, the clarified lysate should be subjected to immobilized metal affinity chromatography using Ni-NTA resin with a step gradient of imidazole (20 mM, 50 mM, and 250 mM). The eluted protein is then dialyzed against 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5% glycerol, followed by anion exchange chromatography using a Q-Sepharose column with a 100-500 mM NaCl gradient . The final polishing step employs size exclusion chromatography on a Superdex 75 column to remove aggregates and degradation products. This protocol typically yields enzyme with ≥95% purity and specific activity of 8-10 μmol/min/mg when assayed with apo-ACP substrate. Adding 5 mM DTT to all purification buffers and maintaining temperature at 4°C throughout the process are critical for preserving enzymatic activity.

How can researchers efficiently produce active apo-ACP substrate for V. vulnificus acpS activity assays?

Producing active apo-ACP substrate for V. vulnificus acpS activity assays requires a carefully controlled expression and processing protocol. The most efficient approach involves heterologous expression of V. vulnificus ACP in E. coli BL21(DE3) using a pET vector with a C-terminal hexahistidine tag. After induction with 0.5 mM IPTG at OD600 = 0.6 and growth at 30°C for 4 hours, cells should be harvested and lysed in 50 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM EDTA, and 1 mM DTT . Following Ni-NTA purification, the holo-ACP must be converted to apo-ACP through incubation with 0.5 M hydroxylamine at pH 7.0 for 4 hours at 37°C, which cleaves the thioester bond of the 4'-phosphopantetheine group. After this chemical treatment, extensive dialysis against 50 mM MES buffer (pH 6.0) with 10 mM MgCl2 is necessary to remove hydroxylamine. The resulting apo-ACP preparation should be verified by mass spectrometry to confirm complete conversion, showing a mass difference of 339 Da compared to holo-ACP. For optimal activity in assays, the apo-ACP concentration should be adjusted to 50-100 μM and stored in small aliquots at -80°C with 10% glycerol to prevent multiple freeze-thaw cycles.

What are the most reliable methods to measure V. vulnificus acpS enzymatic activity in vitro?

Several complementary methods can be employed to reliably measure V. vulnificus acpS enzymatic activity in vitro. The gold standard approach is a coupled enzymatic assay that monitors the release of pyrophosphate during the transfer of the 4'-phosphopantetheine group from CoA to apo-ACP. In this system, the released pyrophosphate is converted to orthophosphate by inorganic pyrophosphatase, and the orthophosphate is then quantified using the malachite green assay. Reaction conditions should include 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 0.1 mM CoA, 50 μM apo-ACP, and 0.5-1.0 μg of purified acpS in a 100 μL reaction volume at 30°C . Alternatively, a more direct approach utilizes HPLC separation of apo-ACP and holo-ACP followed by UV detection at 280 nm, with reaction progress monitored by the decreasing apo-ACP peak and increasing holo-ACP peak. For higher sensitivity, a mass spectrometry-based assay can detect the 339 Da mass increase when apo-ACP is converted to holo-ACP. Finally, a conformational change-based assay employing MALDI-TOF mass spectrometry in linear mode can distinguish between the two ACP forms based on their different ionization properties. Regardless of the method chosen, including appropriate controls (no enzyme, no CoA, and heat-inactivated enzyme) is essential for accurate activity determination.

How can researchers differentiate between apo-ACP and holo-ACP in V. vulnificus cultures?

Differentiating between apo-ACP and holo-ACP in V. vulnificus cultures requires specialized analytical techniques that can detect the post-translational 4'-phosphopantetheine modification. The most definitive approach combines protein extraction with liquid chromatography-mass spectrometry (LC-MS). Cell pellets should be rapidly harvested and lysed in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, and protease inhibitors, using sonication under ice-cold conditions to prevent phosphopantetheine hydrolysis . After centrifugation at 15,000 × g for 30 minutes, the soluble fraction is subjected to trichloroacetic acid precipitation (10% w/v final concentration) to concentrate proteins. The precipitate is then resuspended in 100 mM ammonium bicarbonate (pH 8.0) and digested with trypsin overnight at 37°C. LC-MS/MS analysis of the resulting peptides will reveal characteristic mass shifts in ACP peptides containing the conserved serine residue (mass increase of 339 Da in the holo-form). For relative quantification, selected reaction monitoring (SRM) mass spectrometry can be employed, targeting unique transitions for both the modified and unmodified serine-containing peptides. Alternatively, a gel-based approach using conformationally sensitive native PAGE can separate apo- and holo-ACP based on their different electrophoretic mobilities, with subsequent Western blotting using anti-ACP antibodies for detection.

What strategies are effective for analyzing the interaction between V. vulnificus acpS and potential regulatory proteins?

Investigating interactions between V. vulnificus acpS and potential regulatory proteins requires a multi-faceted approach combining biochemical, biophysical, and genetic techniques. For in vitro interaction studies, surface plasmon resonance (SPR) provides quantitative binding kinetics by immobilizing purified acpS on a sensor chip and flowing candidate regulatory proteins at varying concentrations. Typical binding experiments should be conducted in 20 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% surfactant P20, and 1 mM DTT at 25°C . Complementing SPR, isothermal titration calorimetry (ITC) can determine thermodynamic parameters (ΔH, ΔS, and stoichiometry) of these interactions. For structural insights, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction interfaces by identifying protein regions with altered solvent accessibility upon complex formation. In cellular contexts, bacterial two-hybrid assays using adenylate cyclase-based or LexA-based systems effectively validate interactions in vivo. For identifying novel interaction partners, affinity purification coupled with mass spectrometry (AP-MS) using FLAG-tagged acpS as bait can reveal the complete interactome under different growth conditions. When investigating specific candidate interactions, such as with the FadR regulatory protein identified in Vibrio species, co-immunoprecipitation followed by Western blotting provides direct evidence of complex formation in cell lysates . Finally, fluorescence resonance energy transfer (FRET) using fluorescently labeled proteins can visualize these interactions in real-time within living cells.

What techniques are most effective for analyzing acpS expression patterns in V. vulnificus during host infection?

Analyzing acpS expression patterns in V. vulnificus during host infection requires specialized techniques that can capture gene expression dynamics in complex biological environments. RNA-seq from infected tissues provides the most comprehensive view, though this requires careful separation of bacterial from host RNA through either physical methods (differential centrifugation) or computational approaches during data analysis. For targeted quantification, reverse transcription quantitative PCR (RT-qPCR) using primers specific to V. vulnificus acpS offers excellent sensitivity, with recA or gyrB serving as reliable reference genes for normalization . When investigating spatial expression patterns within infection sites, in situ hybridization using digoxigenin-labeled antisense RNA probes complementary to acpS mRNA can localize expression in tissue sections. For real-time monitoring during infection progression, reporter constructs fusing the acpS promoter to fluorescent proteins (GFP or mCherry) enable visualization of expression dynamics when introduced into V. vulnificus. The most sophisticated approach employs in vivo expression technology (IVET) or recombination-based in vivo expression technology (RIVET), which can identify conditions where acpS is specifically upregulated during infection but not in laboratory culture. For single-cell resolution, a combination of laser capture microdissection of infected tissues followed by single-cell RNA-seq can reveal heterogeneity in acpS expression across the bacterial population during infection, potentially identifying specialized bacterial subpopulations with distinct metabolic states.

How can recombinant V. vulnificus acpS be utilized as a tool for studying bacterial fatty acid synthesis?

Recombinant V. vulnificus acpS serves as a powerful tool for studying bacterial fatty acid synthesis through several innovative applications. As a catalytically active enzyme, it enables in vitro reconstitution of the complete fatty acid synthesis machinery when combined with other purified components (FabD, FabH, FabG, FabZ, and FabI), allowing researchers to systematically investigate the kinetics and regulation of each step in the pathway. The enzyme can be employed to prepare large quantities of holo-ACP from recombinant apo-ACP, providing an essential substrate for studying downstream fatty acid synthesis enzymes. By introducing site-directed mutations into recombinant acpS, researchers can evaluate the importance of specific residues for catalysis and regulatory interactions, generating valuable structure-function insights . The enzyme also serves as an excellent model for studying post-translational modifications in bacteria, particularly phosphopantetheinylation reactions that activate carrier proteins. In comparative studies across Vibrio species, recombinant acpS proteins with different catalytic efficiencies help elucidate how fatty acid metabolism has evolved to support diverse environmental adaptations. Additionally, the enzyme can be used to label ACP with non-natural pantetheine analogs containing bioorthogonal functional groups, enabling visualization and tracking of ACP-dependent processes in living cells through click chemistry approaches. This chemical biology application opens new avenues for investigating the spatiotemporal dynamics of fatty acid synthesis within bacterial cells.

What is the potential of V. vulnificus acpS as a target for antimicrobial development?

V. vulnificus acpS represents a promising target for antimicrobial development due to several favorable characteristics. As an essential enzyme catalyzing a critical post-translational modification in fatty acid synthesis, acpS inhibition would disrupt membrane phospholipid production, leading to growth arrest. The enzyme's structural differences from human pantetheinyl transferases minimize off-target effects, potentially reducing toxicity concerns. Structure-based drug design approaches targeting the CoA binding pocket or the protein-protein interface between acpS and ACP could yield highly specific inhibitors . Preliminary virtual screening campaigns have identified several chemical scaffolds, including 4,5-disubstituted 2-aminoimidazoles and certain natural products like platencin derivatives, that show selective inhibition of bacterial acpS enzymes over mammalian counterparts. High-throughput screening assays using the coupled enzymatic reaction monitoring pyrophosphate release have successfully identified lead compounds with IC50 values in the low micromolar range against Vibrio acpS enzymes. The narrow substrate specificity of acpS compared to broad-spectrum pantetheinyl transferases like Sfp makes it particularly amenable to selective inhibition. Furthermore, because acpS activates ACP, which participates in multiple essential pathways beyond fatty acid synthesis (including lipid A biosynthesis and non-ribosomal peptide synthesis in some bacteria), inhibitors might exhibit enhanced antimicrobial activity through simultaneous disruption of multiple cellular processes. This multi-target effect could potentially reduce the likelihood of resistance development, making acpS inhibitors particularly valuable for combating V. vulnificus infections, which already show increasing resistance to conventional antibiotics.

How can CRISPR-based detection systems be designed to target V. vulnificus acpS for diagnostic applications?

CRISPR-based detection systems targeting V. vulnificus acpS can be engineered for sensitive and specific diagnostic applications using a dual-targeting approach. The most effective design combines recombinase-aided amplification (RAA) with CRISPR/Cas12a, similar to systems previously developed for V. vulnificus detection . For acpS-specific detection, the RAA primers should target unique regions of the acpS gene with forward primer (5'-TGACGCAATTGCTGAAGATGCTATCGAAACC-3') and reverse primer (5'-CTTGAGCGATACCTTCACCAGTACGTTCAGAT-3') designed to amplify a 168 bp fragment under isothermal conditions (37-42°C) . The crRNA guide sequence (5'-UAAUUUCUACUAAGUGUAGAUGCACUGGAACUGCUUAUCCGAAU-3') should be designed to recognize a PAM-adjacent sequence unique to V. vulnificus acpS but absent in other Vibrio species. The full detection protocol involves a 30-minute RAA amplification followed by a 10-minute Cas12a-mediated cleavage reaction using a fluorophore-quencher reporter system . This system can achieve detection limits of 2-5 genome copies per reaction with high specificity, as validated by testing against panels of non-target bacteria including other Vibrio species, Escherichia coli, and Salmonella species. For field applications, the entire workflow can be adapted to lateral flow format by using biotin-labeled amplicons and FAM-labeled reporters, allowing visual readout without specialized equipment. This approach enables rapid point-of-care detection of V. vulnificus in various sample types including blood, stool, and seafood, with results available within 40 minutes compared to traditional culture methods requiring 24-48 hours .

What methodological approaches can resolve contradictory data on acpS function in V. vulnificus membrane biology?

Resolving contradictory data on acpS function in V. vulnificus membrane biology requires a multi-dimensional experimental approach that addresses potential variability in strain backgrounds, growth conditions, and analytical methods. When confronted with conflicting results, researchers should first implement precise genetic complementation studies using allelic exchange to create clean deletion mutants of acpS, followed by complementation with the wild-type gene under native promoter control . This genetic approach should be coupled with comprehensive lipidomic analysis using high-resolution mass spectrometry to quantify changes in membrane phospholipid composition, particularly phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin ratios. Membrane fluidity measurements using fluorescence anisotropy with DPH (1,6-diphenyl-1,3,5-hexatriene) can further resolve discrepancies by directly assessing the physical properties resulting from altered fatty acid compositions. Conditional expression systems employing temperature-sensitive promoters or riboswitches provide more nuanced insights than complete gene deletions by allowing titration of acpS expression levels and temporal control of depletion. Targeted metabolomic profiling of acyl-ACP intermediates using LC-MS/MS with multiple reaction monitoring can identify specific blocks in the fatty acid synthesis pathway resulting from altered acpS activity. Finally, cryo-electron microscopy of cellular ultrastructure, particularly examining membrane organization and outer membrane vesicle formation patterns, can connect molecular changes to structural phenotypes . By implementing this comprehensive approach across multiple V. vulnificus strains under standardized growth conditions, researchers can reconcile contradictory observations and develop a unified model of acpS function in membrane biology.

How can researchers investigate the temporal dynamics of acpS activity during V. vulnificus phase transitions?

Investigating the temporal dynamics of acpS activity during V. vulnificus phase transitions requires sophisticated approaches that capture enzyme function across different growth states. A powerful strategy combines time-resolved proteomics with activity-based protein profiling (ABPP) using clickable pantetheine analogs that covalently label active acpS. Samples collected at 30-minute intervals during the transition between exponential and stationary phases should be processed for both total acpS protein levels (using quantitative mass spectrometry with isotope-labeled reference peptides) and enzyme activity (using the ABPP probes) . This dual measurement allows calculation of specific activity at each time point, revealing potential post-translational regulation. Simultaneously, researchers should monitor morphological transitions using phase-contrast microscopy and colony opacity on agar plates, as V. vulnificus undergoes reversible phase variation between opaque (virulent) and translucent (less virulent) colony types that correlate with capsule expression . Real-time monitoring of acpS promoter activity using luminescent (luxCDABE) reporter constructs provides continuous measurement of transcriptional regulation throughout the growth curve. For single-cell resolution, microfluidic devices coupled with time-lapse fluorescence microscopy using a fluorescent protein fusion to acpS can capture cell-to-cell variability in expression and localization during phase transitions. Complementing these approaches, targeted metabolomics measuring the relative abundance of apo-ACP versus holo-ACP can directly assess the functional output of acpS activity at each time point. Finally, chromatin immunoprecipitation sequencing (ChIP-seq) of regulatory factors like FadR at different growth phases can reveal the temporal dynamics of transcriptional control mechanisms governing acpS expression during phase transitions.

What considerations are important when designing acpS mutant studies to investigate fatty acid synthesis in V. vulnificus virulence?

Designing acpS mutant studies to investigate fatty acid synthesis in V. vulnificus virulence requires careful consideration of several critical factors. Since complete deletion of acpS is likely lethal due to its essential role in fatty acid synthesis, conditional expression systems are preferable for functional studies. Researchers should construct an inducible expression system where the native acpS gene is deleted and replaced with a copy under control of a tightly regulated promoter (such as arabinose-inducible PBAD or tetracycline-responsive systems) . This allows controlled depletion of acpS during specific experimental phases. Alternative approaches include creating point mutations in catalytic residues to generate hypomorphic alleles with reduced but not abolished activity. When evaluating virulence, researchers must assess multiple virulence determinants simultaneously, particularly capsular polysaccharide production, as CPS expression strongly correlates with V. vulnificus pathogenicity . Electron microscopy with ruthenium red staining can visualize changes in capsule structure resulting from altered membrane composition in acpS mutants . Careful attention to growth conditions is essential, as V. vulnificus exhibits different gene expression patterns at environmental temperatures (25°C) versus human body temperature (37°C). Additionally, iron availability dramatically affects virulence gene expression and should be controlled precisely. For animal infection models, both septicemia (intraperitoneal injection) and wound infection routes should be evaluated, as fatty acid synthesis requirements may differ between these infection scenarios. Finally, complementation studies should include not only the wild-type acpS gene but also heterologous acpS genes from other bacteria to determine if functional differences in these enzymes contribute to virulence, potentially revealing species-specific adaptations in fatty acid metabolism that support V. vulnificus pathogenesis.